Esters of acyl carnitines with long-chain aliphatic alcohols and pharmaceutical compositions containing same having antimycotic activity

ABSTRACT

Esters of acyl L-carnitines of general formula (I) ##STR1## wherein R is a straight or branched acyl group having 2 to 16 carbon atoms, in particular isobutyryl and isovaleryl; 
     n is an integer comprised between 7 and 15, particularly 10; and, 
     X- is the anion of a pharmacologically acceptable acid are endowed with potent antimycotic activity particularly against yeast like fungi, such as Candida albicans, the aetiologic agent of candidiasis and against filamentous fungi, such as Aspergillus fumigatus, the aetiologic agent of aspergillosis. Orally or parenterally administrable or topically applicable pharmaceutical compositions comprise an ester of formula (I) as active ingredient.

This is a division, of application Ser. No. 08/250,108, filed on May 26,1994, now U.S. Pat. No. 5,519,056, which is a continuation ofapplication Ser. No. 08/000,714, filed on Jan. 5, 1993, now abandoned.

The present invention relates to esters of acyl L-carnitines withlong-chain aliphatic alcohols, of general formula (I) ##STR2## wherein Ris a straight or branched saturated carboxylic acyl group having from 2to 16, preferably from 4 to 12, carbon atoms, in particular isobutyryland isovaleryl;

n is an integer from 7 to 15, particularly 10, and

X- is the anion of a pharmacologically acceptable acid.

Among the esters of formula (I) isovaleryl L-carnitine undecil ester andisobutyryl L-carnitine undecil ester are particularly preferred.

Therefore, the present invention also relates to orally or parenterallyadministrable or topically applicable pharmaceutical compositions usefulfor the treatment of infections such as candidiasis, cryptococcosis andaspergillosis sustained by pathogenic fungi.

Crytpococcosis is an infection of the respiratory tract, generallyasymptomatic and benign which may provoke a systemic spread and seriouscephalomeningitis in both wasted and even healthy subjects. The diseaseis due to the fungus Cryptococcus neoformans (now known asFilobasidiella neoformans) widely distributed in nature, which can betransmitted to humans via inhalation.

Candidiasis encompasses a wide range of mycoses varying from commoninfections of the skin and mucous membranes to life-threatening systemicinfections. These infections are due to some strains belonging to thegenus Candida, particularly C. albicans.

C. albicans is one of the most widely occurring constituents of themicrobial flora of various mucosas such as that of vagina, mouth anddigestive tract, where under conditions particularly unfavourable to thehost but optimal for the fungus growth (such as debilitating affections,prolonged administration of antibiotics and corticosteroids, etc.) itmay bring about life-threatening pathologies.

The main affections induced by this fungus are: vulvovaginitis,dissemination to the lungs (pneumonia) and other organs such as kidney,spleen, eyes, meninges, liver, intestine and endocardium, ascomplication of preexisting diseases; thrush frequently occurring innewborns and young adults (characterized by creamy white patches on thebuccal mucosa); cutaneous candidiasis and onychomycosis.

Aspergillosis, an infectious disease caused by the inhalation of sporesof the genus Aspergillus (fumigatus, fiavus), is a serious and invasivepulmonary infection brought about by the growth of a fungal colony whichfrequently fills preexisting cavities, such as pulmonary abscesses.

Aspergillus spp (e.g. A. niger) may also cause otomycosis. Seriousintoxications (mycotoxicosis) in animals, fed on contaminated fodder,are caused by the toxins (aflatoxins) produced by Aspegillus flavus.Aspergillus flavus is suspected to cause cirrhosis and liver cancer inhumans, in areas wherein foodstuffs are likely to be contaminated byaflatoxins.

The esters of formula (I) may be prepared following two distinctsynthesis processes. The first process (illustrated in the synthesisscheme 1) comprises the steps consisting of:

(a) halogenating an acyl L-carnitine with a halogenating agent, such asthionyl chloride and oxalyl chloride (molar ratio comprised between 1:1and 1:4) in an anhydrous organic inert solvent such as acetonitrile ormethylene chloride at a temperature comprised between 0° C. and 30° C.for 1-4 hours, concentrating the raw reaction product and using it inthe following step;

(b) dissolving the acid chloride of step (a) in an anhydrous organicinert solvent such as acetonitrile or methylene chloride and adding thealcohol diluted in the same solvent at a ratio comprised between 1:1 and1:2, at a temperatures comprised between 0° C. and 30° C. for 2-10hours, concentrating the solution and, if needed, purifying the compoundby chromatography on silica gel; and

(c) eluting the product dissolved in water or in an organic solvent on astrongly basic ion exchange resin such as Amberlite IRA 402 or on aweakly basic ion exchange resin such as Amberlist A 21, activated withthe desired HX acid and isolating the end product by lyophilization orconcentration.

The second process (illustrated in the synthesis scheme 2) comprise thesteps consisting of:

(a') reacting carnitine or an acyl carnitine inner salt with therelevant alkyl halogcnide (preferably bromide or iodide) in an organicanhydrous inert solvent at a temperature comprised between 30° C. and60° C. for 8-24 hours and then isolating the resulting compound byconcentration;

(b') acylating the ester obtained in step (a') with the desired acidchloride by known techniques, in case the starting compound in step (a')is carnitine;

(c') eluting an aqueous or alcoholic solution of the compound of step(a') or (b') on an ion exchange resin, such as Amberlite IRA 402 orAmberlist A 21 activated with the desired HX acid.

The anion X- of the pharmacologically acceptable acid is preferablyselected from chloride; bromide; iodide; aspartate, particularly acidasparatate; citrate, particularly acid citrate; tartrate; phosphate,particularly acid phosphate; fumarate, particularly acid fumarate;glycerophosphate; glucosephosphate; lactate; maleate, particularly acidmaleate; orotate; oxalate, particularly acid oxalate; sulphate,particularly acid sulphate; trichloroacetate; trifluoroacetate andmethansulphonate. ##STR3##

EXAMPLE 1 PREPARATION OF ISOVALERYL-L-CARNITINE UNDECYL ESTER CHLORIDE(ST 722) Step A Preparation of isovaleryl-L-carnitine chloride acidchloride.

Isovaleryl-L-carnitine chloride (30 g; 0.106 moles) was suspended in 100ml anhydrous CH₂ Cl₂.

The mixture was cooled at 0° C. and oxalyl chloride (13 ml; 0.15 moles)diluted in 15 ml anhydrous CH₂ Cl₂ was slowly added under stirring.

After 30 minutes at room temperature, a further amount of oxalylchloride (19 ml; 0.21 moles) diluted in 10 ml anhydrous CH₂ Cl₂ wasadded.

The resulting solution was kept under stirring for 2 hours at roomtemperature, then concentrated under vacuum.

The residue thus obtained was washed twice with anhydrous CH₂ Cl₂ andconcentrated under vacuum.

The raw product thus obtained was used as such in the next reaction.

Step B: Preparation of isovaleryl-L-carnitine undecyl ester chloride (ST722).

The acid chloride previously prepared (0.106 moles) was dissolved inanhydrous CH₂ Cl₂ (40 ml).

The solution was cooled at 0° C. and undecylic acid (35 ml; 0.168 moles)diluted in 35 ml CH₂ Cl₂ was added in a nitrogen atmosphere.

The solution was kept under stirring at room temperature for 2 hours andthen concentrated under vacuum until an oily residue was obtained.

The raw reaction mixture was chromatographed on a silica gel columnbuffered with 2% Na₂ HPO₄, eluting with CH₂ Cl₂ till complete elution ofundecylic alcohol and then with CH₂ Cl₂ -MeOH 9:1 till complete elutionof the compound.

The pooled fractions were concentrated and gave 28 g of the titlecompound; Yield 60%.

    [α].sub.D.sup.25 =-10.5 (c=1% H.sub.2 O))

    ______________________________________                                        Elementary analysis for C.sub.23 H.sub.46 ClNO.sub.4                                        C %  H %      Cl %    N %                                       ______________________________________                                        Calculated (anhydrous)                                                                        63.35  10.63    8.13  3.21                                    Found           60.87   0.88    8.14  3.29                                    ______________________________________                                    

H₂ O 2.4%

HPLC

Column: Spherisorb Cl 5 μm

t.: 50° C.

Eluant: CH₃ OH/KH₂ PO₄ 50 mM (65:35)

Flow rate: 1 ml/min.

Retention time: 14.82 min

NMR CDCl3 δ5.5 (1H,m,--CH--); 4.2-3.8(4H,m,N+CH₂ ; OCH₂);3.3(9H,S,(CH₃)₃ N+); 2.8(2H,m,CH₂ COO); 2.2(2H, m,OCOCH₂); 1.6-1.0(22H,m##STR4## (CH₂)₉ CH₃); 0.8(6H,d) ##STR5##

EXAMPLE 2 PREPARATION OF ISOBUTYRYL-L-CARNITINE UNDECYL ESTER CHLORIDE(ST 712)

The compound was prepared as described in example 1, substitutingisobutyryl L-carnitine chloride for isovaleryl L-carnitine chloride.Yield 55%.

    [α].sub.D.sup.25 =-15.8 (c=1% H.sub.2 O)

    ______________________________________                                        Elementary analysis for C.sub.22 H.sub.44 O.sub.4 NCl                                       C %  H %      Cl %    N %                                       ______________________________________                                        Calculated (anhydrous)                                                                        62.61  10.51    3.32  8.40                                    Found           61.77  10.67    3.29  8.17                                    ______________________________________                                    

H₂ O 0.8%

HPLC

Column: Spherisorb Cl (4.6 mm)

eluant CH3OH-KH₂ PO₄ 50 mM 60-40

Flow rate: 1 ml/min

Retention time: 14,75 min

NMR CDCl₃ δ5.5 (1H,m); ##STR6## 4.2-3.8(4H,m,N+CH₂ -;OCH₂); 3.3(9H,s,(CH₃)₃ N+); 2.8(2H,m,CH₂ COO); 2.5 (1H,m,COCH); 1.5-0.9 (27H,m##STR7## (CH₂)₉ -CH₃

EXAMPLES 3-19

The compounds of Examples 3-19 were prepared following the procedures ofthe previous examples, as apparent to any average expert in organicsynthesis. The physico-chemical characteristics of the compounds aresummarized in the following table.

    __________________________________________________________________________    Ex                    E.A. found         m.p. HPLC                            code R     n X.sup.-                                                                          [α].sub.D.sup.25                                                              C   H   N   Cl  H.sub.2 O                                                                        °C.                                                                         Rt min                                                                            NMR δ                 __________________________________________________________________________    3    octanoyl                                                                            10                                                                              Cl.sup.-                                                                         -10,7 C.sub.26 H.sub.52 NO.sub.4 Cl                                                                    103-105                                                                            10,28.sup.a                                                                       5,7(1H, m, CHO);            ST 1000         (c =1%                                                                              62,84%                                                                            10,7%                                                                             3,03%                                                                             6,96%                                                                             3,2%        4,3-4,0(4H, m, CH.sub.2                                                       N.sup.+ ;                                   CHCl.sub.3)                       OCH.sub.2);                                                                   3,5(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,3(2H, t,                                                                    OCOCH.sub.2);                                                                 1,8(4H, m, 2CH.sub.2);                                                        1,6(4H, m; 2CH.sub.2);                                                        1,3(20H, broad,                                                               10CH.sub.2);                                                                  0,9(6H, 2t,                                                                   2CH.sub.3).CDCl.sub.3       4    undecanoyl                                                                          10                                                                              Cl.sup.-                                                                         -14   C.sub.29 H.sub.58 NO.sub.4 Cl                                                                    133-135                                                                            16,32.sup.b                                                                       5,6(1H, m, CHO);             ST 982         (c = 0,5%                                                                           66,65%                                                                            11,72%                                                                            2,70%                                                                             6,93%                                                                             0,4%        4,1(2H, t, OCH.sub.2);                      MetOH)                            3,9-3,7(2H, m, CH.sub.2                                                       N.sup.+);                                                                     3,3(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     2,8(2H, dd, CH.sub.2                                                          COO);                                                                         2,4(2H, t,                                                                    OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2);                                                        1,3(30H, broad,                                                               15CH.sub.2);                                                                  0,9(6H, t,                                                                    2CH.sub.3).CD.sub.3 OD      5    palmitoyl                                                                           10                                                                              Cl.sup.-                                                                         -14   C.sub.34 H.sub.68 NO.sub.4 Cl                                                                    158-159                                                                            10,5.sup.c                                                                        5,6(1H, m, CHO);            ST983           (c = 1%                                                                             69,42%                                                                            11,84%                                                                            2,36%                                                                             5,96%                                                                             0,8%        4,1(2H, t, OCH.sub.2);                      MetOH)                            3,9-3,7(2H, m, CH.sub.2                                                       N.sup.+);                                                                     3,2(9H, s,                                                                    (CH.sub.3).sub.3)N.sup.+                                                      );                                                                            2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,4(2H, t,                                                                    OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2);                                                        1,3(40H, broad,                                                               20CH.sub.2);                                                                  0,9(6H, t,                                                                    2CH.sub.3).CD.sub.3 OD      6    isocaproyl                                                                          10                                                                              Cl.sup.-                                                                         -13,12                                                                              C.sub.24 H.sub.48 NO.sub.4 Cl                                                                    oil/not                                                                            8,45.sup.d                                                                        5,7(1H, m, CHO);            ST 1034         (c = 0,8%                                                                           61,28%                                                                            11,10%                                                                            3,12%                                                                             9,01%                                                                             1,9%                                                                             deter-   4,1(2H, t, OCH.sub.2);                      H.sub.2 O)               mined    4,0-3,7(2H, m, CH.sub.2                                                       N.sup.+);                                                                     3,2(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     3,0-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,6-2,3(2H, m,                                                                OCOCH.sub.2);                                                                 1,7-1,4(5H, m,                                                                2CH.sub.2,                                                                    --CH);                                                                        1,3(16H, broad,                                                               8CH.sub.2);                                                                   0,9(6H, d,                                                                    (CH.sub.3).sub.2);                                                            0,8(3H, t,                                                                    CH.sub.3).D.sub.2 O         7    heptanoyl                                                                           10                                                                              Cl.sup.-                                                                         -12,1 C.sub.25 H.sub.49 NO.sub.4 Cl                                                                    not  9,35.sup.d                                                                        5,7(1H, m, CHO);            ST 1036         (c = 1%                                                                             64,35%                                                                            12,55%                                                                            3,09%                                                                             6,68%                                                                             1,3%                                                                             deter-   4,1(2H, t, OCH.sub.2);                      H.sub.2 O)               mined    4,0-3,7(2H, m, CH.sub.2                                                       N.sup.+);                                                                     3,2(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     3,0-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,5-2,3(2H, m,                                                                COCH.sub.2);                                                                  1,6(4H, m, 2CH.sub.2);                                                        1,3(22H, m,                                                                   11CH.sub.2);                                                                  0,9-0,8(6H, 2t,                                                               2CH.sub.3).D.sub.2 O        8    heptanoyl                                                                           12                                                                              Cl.sup.-                                                                         -10,3 C.sub.27 H.sub.54 NO.sub.4 Cl                                                                    dec. 9,13.sup.e                                                                        5,7(1H, m, CHO);            ST 1050         (c = 0,7%                                                                           65,26%                                                                            11,62%                                                                            2,87%                                                                             6,70%                                                                             0,3%                                                                             150-160  4,3-4,0(4H, m, CH.sub.2                                                       N.sup.+ ;                                   CHCl.sub.3)                       OCH.sub.2);                                                                   3,5(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,3(2H, m,                                                                    OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2);                                                        1,3(26H, m,                                                                   13CH.sub.2);                                                                  0,9(6H, 2t,                                                                   2CH.sub.3).CDCl.sub.3       9    2-methyl                                                                            12                                                                              Cl.sup.-                                                                         -8,8  C.sub.27 H.sub.54 NO.sub.4 Cl                                                                    not  28,03.sup.b                                                                       5,7(1H, m, CHO);            ST 1051                                                                            hexanoyl   (c = 1%                                                                             65,06%                                                                            11,32%                                                                            2,91%                                                                             6,93%                                                                             0,4%                                                                             deter-                                                                                 4,3-4,0(4H, m, CH.sub.2                                                       N.sup.+ ;                                   CHCl.sub.3)              mined    OCH.sub.2);                                                                   3,5(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,4(1H, m, CH);                                                               1,6(2H, m, CH.sub.2);                                                         1,3(26H, m,                                                                   13CH.sub.2);                                                                  1,1(3H, m,                                                                    CHCH.sub.3);                                                                  0,9(6H, 2t,                                                                   2CH.sub.3).CDCl.sub.3       10   isovaleryl                                                                          12                                                                              Cl.sup.-                                                                         -11,8 C.sub.25 H.sub.50 NO.sub.4 Cl                                                                    dec. 9,39.sup.d                                                                        5,7(1H, m, CHO);            ST 1033         (c = 1%                                                                             63,73%                                                                            12,50%                                                                            3,17%                                                                             7,03%                                                                             1,2%                                                                             150      4,1(2H, m, OCH.sub.2);                      H.sub.2 O)                        4,0-3,7(2H, m, CH.sub.2                                                       N.sup.+);                                                                     3,2(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     3,0-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,3(2H, m,                                                                    OCOCH.sub.2);                                                                 2,1(1H, m, CH.sub.2                                                           CH);                                                                          1,6(2H, m, CH.sub.2);                                                         1,3(20H, broad,                                                               11CH.sub.2);                                                                  0,9(6H, dd,                                                                   CH(CH.sub.3).sub.2);                                                          0,8(3H, t,                                                                    CH.sub.3).D.sub.2 O         11   hexanoyl                                                                            12                                                                               Cl.sup.-                                                                        -10,7 C.sub.26 H.sub.52 NO.sub.4 Cl                                                                    dec. 14,86.sup.c                                                                       5,7(1H, m, CHO);            ST 1052         (c = 1%                                                                             65,01%                                                                            11,87%                                                                            2,93%                                                                             7,14%                                                                             1,2%                                                                             120-130  4,3-4,0(4H, m, CH.sub.2                                                       N.sup.+ ;                                   CHCl.sub.3)                       OCH.sub.2);                                                                   3,5(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     2,9-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,3(2H, t,                                                                    OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2);                                                        1,3(24H, broad,                                                               12CH.sub.2);                                                                  0,9(6H, m,                                                                    2CH.sub.3).CDCl.sub.3       12   octanoyl                                                                            12                                                                              Cl.sup.-                                                                         -9,8  C.sub.28 H.sub.56 NO.sub.4 Cl                                                                    dec. 14,71.sup.c                                                                       5,7(1H, m, CHO);            ST 1053         (c = 1%                                                                             66,46%                                                                            11,93%                                                                            2,71%                                                                             6,93%                                                                             0,7%                                                                             150-160  4,3-4,0(4H, m, CH.sub.2                                                       N.sup.+ ;                                   CHCl.sub.3)                       OCH.sub.2);                                                                   3,5(9H, s,                                                                    (CH.sub.3).sub.3                                                              N.sup.+);                                                                     2,9-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,3(2H, m, OCOCH.sub.                                                         2);                                                                           1,6(4H, m, 2CH.sub.2);                                                        1,3(28H, broad,                                                               14CH.sub.2);                                                                  0,9(6H, m,                                                                    2CH.sub.3).CDCl.sub.3       13   isovaleryl                                                                          11                                                                              Cl.sup.-                                                                         -12,2 C.sub.24 H.sub.48 NO.sub.4 Cl                                                                    dec. 12,65.sup.a                                                                       5,7(1H, m, CHO);            ST 1037         (c = 1%                                                                             63,46%                                                                            12,26%                                                                            3,15%                                                                             7,81%                                                                             1,0%                                                                             150-160  4,4-4,0(4H, m, N.sup.+                                                        CH.sub.2 ;                                  H.sub.2 O)                        OCH.sub.2)                                                                    3,5(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,2(2H, m,                                                                    OCOCH.sub.2);                                                                 2,0(1H, m,                                                                    CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2);                                                         1,2(18H, broad,                                                               9(CH.sub.2);                                                                  0,9-0,8(9H, d+t,                                                              CH.sub.3 ;                                                                    (CH.sub.3).sub.2).CDCl.s                                                      ub.3                        14   isobutyryl                                                                          11                                                                              Cl.sup.-                                                                         -14,5 C.sub.23 H.sub.46 NO.sub.4 Cl                                                                    dec. 14,0.sup.a                                                                        5,7(1H, m, CHO);            ST 1038         (c = 1%                                                                             62,90%                                                                            11,47%                                                                            3,27%                                                                             7,86%                                                                             0,4%                                                                             150- 155 4,4-4,0(4H, m, N.sup.+                                                        CH.sub.2 ;                                  H.sub.2 O)                        OCH.sub.2);                                                                   3,5(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            2,9-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,6-2,5(1H, m,                                                                CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2);                                                         1,3(18H, broad,                                                               9CH.sub.2);                                                                   1,1(6H, d,                                                                    CH(CH.sub.3).sub.2);                                                          0,8(3H, t, CH3).CDCl3       15   heptanoyl                                                                           11                                                                              Cl.sup.-                                                                         -12,7 C.sub.26 H.sub.52 NO.sub.4 Cl                                                                    not  10,47.sup.f                                                                       5,7(1H, m, CHO);            ST 1060         (c = 1%                                                                             67,00%                                                                            12,12%                                                                            2,41%                                                                             6,60%                                                                             0,6%                                                                             deter-   4,4-4,0(4H, m, N.sup.+                                                        CH.sub.2 ;                                  MetOH)                   mined    OCH.sub.2);                                                                   3,5(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,4(2H, t,                                                                    COOCH.sub.2)                                                                  1,6(2H, m, CH.sub.2);                                                         1,3(26H, broad,                                                               13CH.sub.2);                                                                  0,9(6H, 2t, 2CH.sub.3)      16   isovaleryl                                                                          15                                                                              Cl.sup.-                                                                         -12,6 C.sub.28 H.sub.56 NO.sub.4 Cl                                                                    not  12,12.sup.a                                                                       5,7(1H, m, CHO);            ST 1001         (c = 0,5%                                                                           65,30%                                                                            11,11%                                                                            2,68%                                                                             7,57%                                                                             0,5%                                                                             deter-   4,4-4,0(4H, m, N.sup.+                                                        CH.sub.2 ;                                  H.sub.2 O)               mined    OCH.sub.2);                                                                   3,5(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,2(3H, m,                                                                    CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2);                                                         1,3(26H, broad,                                                               13CH.sub.2);                                                                  1,0-0,9(9H, d+t,                                                              CH.sub.3,CH(CH.sub.3).su                                                      b.2).CDCl.sub.3             17   isovaleryl                                                                          10                                                                              tar-                                                                             -1,9  C.sub.27 H.sub.51 NO.sub.10                                                                      not  13,74.sup.a                                                                       5,7(1H, m, CHO);            ST 1018      trate                                                                            (c = 1%                                                                             56,87%                                                                             9,78%                                                                            2,44%     4%                                                                             deter-   4,5(2H, s, 2CHOH);                       acid                                                                             H.sub.2 O)               mined    4,1-3,6(4H, m, N.sup.+                                                        CH.sub.2 ;                                                                    OCH.sub.2);                                                                   3,2(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            3,0-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,4-2,2(2H, m,                                                                OCOCH.sub.2);                                                                 2,1-2,0(1H, m,                                                                CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2);                                                         1,3(16H, broad,                                                               8CH.sub.2);                                                                   0,9(6H, d,                                                                    CH(CH.sub.3).sub.2);                                                          0,8(3H, t,                                                                    CH.sub.3).D.sub.2 O         18   isovaleryl                                                                          10                                                                              fu-                                                                              -13,3%                                                                              C.sub.27 H.sub.49 NO.sub.8                                                                       dec. 13,99.sup.a                                                                       6,6(2H, s, CH═CH);      ST 1017      ma-                                                                              (c = 1%                                                                             62,23%                                                                             9,90%                                                                            2,54%   0,7%                                                                             120      5,7(1H, m, CHO);                         rate                                                                             H.sub.2 O)                        4,1-3,7(4H, m, N.sup.+                                                        CH.sub.2 ;                               acid                                 OCH.sub.2);                                                                   3,2(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            3,0-2,7(2H, m, CH.sub.2                                                       COO);                                                                         2,4-2,2(2H, m,                                                                OCOCH.sub.2);                                                                 2,0(1H, m,                                                                    CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2);                                                         1,3(16H, broad,                                                               8CH.sub.2);                                                                   0,9(6H, t,                                                                    CH(CH.sub.3).sub.2);                                                          0,8(3H, t,                                                                    CH.sub.3).D.sub.2 O         19   isovaleryl                                                                           8                                                                              Cl.sup.-                                                                         -17,4%                                                                              C.sub.20 H.sub.40 ClNO.sub.4                                                                     not  5,75.sup.b                                                                        5,7(1H, m, CHO);            ST 1032         (c = 1%                                                                             59,93%                                                                            10,25%                                                                            3,49%                                                                             8,84%                                                                             1,7%                                                                             deter-   3,8-4,1(4H, m, N.sup.+                                                        CH.sub.2 ;                                  H.sub.2 O)               mined    OCH.sub.2);                                                                   3,3(9H, s, N.sup.+                                                            (CH.sub.3).sub.3);                                                            2,8(2H, m, CH.sub.2                                                           COO);                                                                         2,3(2H, m,                                                                    COCH.sub.2);                                                                  2,1(1H, m,                                                                    CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2);                                                         1,3(10H, m, broad);                                                           1,0-0,9(9H, d+t,                                                              CH.sub.3 CH(CH.sub.3).su                                                      b.2.D.sub.2 O               __________________________________________________________________________    .sup.a Column:                                                                       Nucleosil-SA (5μ) 1,2 mm, i.d. 4,0 mm                               T:     40° C.                                                          mobile phase:                                                                        (NH.sub.4).sub.2 HPO.sub.4 50 mM/CH.sub.3 CN 1:1 pH4 con H.sub.3              PO.sub.4                                                               flow:  0,75 ml/min                                                            .sup.b Column:                                                                       Spherisorb-Cl (5μ) 1,2 mm i.d. 4,6 mm                               T:     50° C.                                                          mobile phase:                                                                        CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 60:40                              flow:  0,5 ml/min                                                             .sup.c Column:                                                                       Spherisorb-Cl (5μ) 1,2 mm i.d. 4,6 mm                               T:     50° C.                                                          mobile phase:                                                                        CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 70:30 pH 3,9 con H.sub.3                  PO.sub.4                                                               flow:  0,5 ml/min                                                             .sup. d Column:                                                                      Spherisorb-Cl (5μ) 1,2 mm i.d. 4,6 mm                               T:     40° C.                                                          mobile phase:                                                                        CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 65:35 pH 4,5 con H.sub.3                  PO.sub.4                                                               flow:  0,5 ml/min                                                             .sup.e Column:                                                                       Nucleosil-SA (5μ) 1,2 mm, i.d. 4,0 mm                               T:     30° C.                                                          mobile phase:                                                                        (NH.sub.4).sub.2 HPO.sub.4 50 mM/CH.sub.3 CN 65:35 pH 3,5 con                 H.sub.3 PO.sub.4                                                       flow:  0,75 ml/min                                                            .sup.f Column:                                                                       Spherisorb-Cl (5μ) 1,2 mm i.d. 4,6 mm                               T:     40° C.                                                          mobile phase:                                                                        CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 65:35 pH 4,5 con H.sub.3                  PO.sub.4                                                               flow:  1 ml/min                                                           

ASSESSMENT OF BEHAVIOUR AND MORTALITY IN MICE

The assessment of normal behaviour in mice was carried out following S.Irwin's method (Psychopharmacologia, 13, 222 [1968]). This method allowsalterations in some behavioural, neurophysiologic and neurovegetativeparameters to be detected, which are directly observable by theresearcher. The study was conducted using male Crl:(CD-1)(ICR)BR mice(Charles River-Italy) weighing 22-25 g. following oral administration ofthe compounds suspended in carboxymethylcellulose (0.5% by weight in H₂O) to groups of 4 animals/dose.

The animals were continously kept under observation for five hoursfollowing treatment and twice a day in the subsequent five days.Mortality was also observed during the overall test period.

Assessment of behaviour and mortality in mice.

    ______________________________________                                        Compound  dose      Symptoms      Mortality                                   ______________________________________                                        ST 712    1000      NONE          O/4                                         ST 722    1000      salivation, diarrhoea                                                                       O/4                                         ______________________________________                                    

IMMUNOTOXICOLOGICAL STUDIES

Some immunotoxicological results after oral ST 722 administration inmice are described hereinafter:

Test 1: Evaluation of the "in vitro-ex vivo" effect of repeated oraladministrations of ST 722 on the primary antibody production (Jernetest) in the spleen of SRBC (Sheep Red Blood Cells) immunized mice.

Experimental procedure

Male B₆ D2F₁ mice (C. River) aged 8 weeks (6 animals each group) wereutilized.

The substance (ST 722) was administered per os at the dose of 100mg/kg/day from day -2 through day +2 (immunization at day 0). Theanimals were immunized by intraperitoneal route with a concentration of1.0×10⁸ cells/mouse in 0.2 ml of sterile saline.

Five days later, spleens to be submitted to teasing process were removedfrom the animals sacrificed by cervical dislocation. Afterstandardization at 1.0×10⁷ cells/ml. the splenocytes (0.1 ml) were mixedwith warm agar-Hank's (2 ml) and 10% SRBC in PBS (0.2 ml) and seeded inPetri dishes (samples tested in triplicates) and incubated at 37° C. for60 min.

After addition of complement (2 ml of guinea pig serum diluted 1:10 inTris buffer), samples were further incubated at 37° C. for 30 min. Toblock the haemolysis reaction, the Petri dishes were cooled at 4° C. andthe haemolysis plaques were counted.

The antibody response to SRBC is expressed as number of plaque formingcells (PFC) per 1.0×10⁶ splenocytes as well as per spleen.

Results

The results indicated that the repeated (5 consecutive days) oraladministrations of ST 722 did not cause a statistically significantmodification in PFC number after the antigenic challenge (Table 1).These data exclude the existence of an immunotoxic effect on theantibody-producer B lymphocytes.

The weight of the lymphold organs (spleen and thymus) did not showvalues relating to a toxic effect as well (Tab. 1 ).

                                      TABLE 1                                     __________________________________________________________________________    Primary antibody production (Jerne test).                                     Evaluation of the number of PFC (x ± S.E.) in the spleen                   of mice immunized with SRBC and treated per os with ST 722                    at the dose of 100 mg/kg/day from day -2 through day +2                       (immunization at day 0).                                                            Body weight                                                                          Spleen weight                                                                         Thymus weight                                                                         PFC/10.sup.6                                                                       PFC/                                        Treatment                                                                           (g)    (mg)    (mg)    cells                                                                              spleen                                      __________________________________________________________________________    Control                                                                             25.33 ± 0.38                                                                      95.50 ± 3.19                                                                       64.00 ± 1.51                                                                       202 ± 32                                                                        34228 ± 5064                             ST 722                                                                              25.12 ± 0.77                                                                      94.33 ± 4.12                                                                       59.50 ± 3.62                                                                       261 ± 34                                                                        44440 ± 6087                             __________________________________________________________________________

Test 2: Evaluation of the effect of repeated oral administrations of ST722 on the weight of murine lymphoid organs (spleen and thymus).

Experimental procedure

Male B₆ D₂ F₁ mice (C. River) aged 7 weeks (7-8 animals each group) wereorally treated with the substance ST 722 at the dose of 100 mg/kg/dayfor 7 consecutive days. Twenty-four hours after the last administration,the animals were sacrificed, the organs removed and weighed.

Results

The performed treatment did not provoke any immunotoxic effect on theparameters examined (Table 2).

                  TABLE 2                                                         ______________________________________                                        Weight (x ± S.E.) of murine lymphoid organs                                after repeated treatment oral of the animals with the                         substance ST 722 (100 mg/kg/day for 7 consecutive days).                              Body weight.sup.a                                                                          Spleen weight.sup.a                                                                       Thymus weight.sup.a                          Treatment                                                                             (g)          (mg)        (mg)                                         ______________________________________                                        Control 24.00 ± 0.68                                                                            79.63 ± 3.31                                                                           51.88 ± 2.72                              ST 722  22.74 ± 0.37                                                                            77.88 ± 2.38                                                                           52.88 ± 3.20                              ______________________________________                                         .sup.a = mean value (x ± E.S.) of 7-8 samples.                        

Test 3: Evaluation of the effect of repeated oral administrations of ST722 on the body, spleen and thymus weight, and on the splenocyteconcentration in mice.

Experimental procedure

Male B₆ D₂ F₁ mice (C. River) aged 10 weeks (5 animals each group) wereorally treated with the substance ST 722 at the dose of 100 mg/kg/dayfor 5 consecutive days. Twenty-four hours after the last administration,the animals were sacrificed, the organs removed and weighed, and thesplenocyte number determined.

Results

The results, reported in Table 3, showed a lack of specific immunotoxiceffects on the considered parameters following the scheduled ST 722treatment.

                                      TABLE 3                                     __________________________________________________________________________    Weight of lymphoid organs and spleen concentration after                      repeated oral treatments of mice with the substance ST 722                    (100 mg/kg/day for 5 consecutive days).                                              Body weight.sup.a                                                                    Spleen weight.sup.a                                                                   Thymus weight.sup.a                                                                   Splenoc. number.sup.b                           Treatment                                                                            (g)    (mg)    (mg)    (× 10.sup.-7)                             __________________________________________________________________________    Control                                                                              27.70 ± 0.48                                                                      75.75 ± 2.36                                                                       41.75 ± 3.59                                                                       7.90                                            ST 722 26.50 ± 0.53                                                                      73.25 ± 3.84                                                                       38.80 ± 3.01                                                                       7.85                                            __________________________________________________________________________     .sup.a = mean value (x ± S.E.) of 5 samples.                               .sup.b = value from 5 pooled samples.                                    

Test 4: Evaluation of the effect of repeated oral administrations of ST722 on the peritoneal macrophage number in mice.

Experimental procedure

Male B₆ D₂ F₁ mice (C. River) aged 10 weeks (6 animals each group) wereorally treated with the substance ST 722 at the dose of 100 mg/kg/dayfor 5 consecutive days. Twenty-four hours after the last administration,the animals were sacrificed, the peritoneal exudate cells (PEC)collected and the macrophage number determined.

Results

No toxic effect has been observed in PEC macrophage population; on thecontrary, we measured an increase of about 60% in the peritonealmacrophages number of mice treated with the substance ST 722 (Table 4).

                  TABLE 4                                                         ______________________________________                                        Peritoneal macrophage (Mo) number in mice treated with ST 722                 (100 mg/kg/day for 5 consecutive days).                                                          PEC/              PEC Mo/                                          Body weight                                                                              mouse.sup.b                                                                             PEC Mo.sup.b                                                                          mouse.sup.b                              Treatment                                                                             (g).sup.a  (× 10.sup.-6)                                                                     (%)     (× 10.sup.-6)                      ______________________________________                                        Control 28.07 ± 0.63                                                                          2.14      53      1.13                                     ST 722  28.13 ± 1.30                                                                          2.86      63      1.80                                     ______________________________________                                         .sup.a = mean value (± S.E.) of 6 animals.                                 .sup.b = Value from 6 pooled samples.                                    

Evaluation of the Minimal Inhibitory Concentration (MIC) of 16 newsubstances for yeast and yeast-like strains. Experimental procedure

The following strains were used (the number of strains is in brackets):Candida (13); Criptococcus (2); Saccharormyces (1); Torulopsis (1).

The tested substances were: ST 712, ST 722, ST 982, ST 983, ST 1000, ST1001, ST. 1032, ST 1033, ST 1034, ST 1036, ST 1037, ST 1038, ST 1050, ST1051, ST 1052, ST 1053.

Yeast Nitrogen Base supplemented with 0.15% asparagine and 1.0% glucose(as carbon source) was the medium utilized in the assays.

This medium was buffered with 0.165 M 2-(N-morpholino) propane sulfonicacid (MOPS), a non-chelating buffer.

The test was performed following the standard microdilution procedureand the inoculum suggestions of Pfaller (PFALLER M., RINALDI M. G.,GALGIANI J. N., BARTLETT M. S., BODY B. A., ESPINELINGROFF A., FROMTLINGR. A., HALL G. S., HUGHES G. E., ODDS F. C. and SUGAR A. M., 1990.Collaborative investigation of variables in susceptibility testing ofyeasts. Antimicrob, Agents Chemother, Sep. 34, 9: 1648-1654) with COOK'smodifications (COOK R., Mc INTYRE K. A. and GALGIANI J. M.,1990. Effectof incubation temperature, inoculurn size, and medium on agreement ofmacro-and microdilution broth susceptibility test, results for yeasts.Antimicrob. Agents Cheroother. Aug. 34.8:1542-1545).

The inoculum was prepared from an overnight broth culture (a 48-hourculture only for Cryptococcus) in Sabouraud broth and appropriatelydiluted to yield a final suspension containing approximately 5.0×10⁶Colony Forming Units per ml (CFU/ml), when compared to the Mc Farland'sstandard control (BRAY W. E., Clinical Laboratory Methods, 5th Ed. C. V.MOSBY, St. Louis, Mo, 1957).

After further diluting (1:50), a 0.150-μl volume of such fungalsuspensions and an equal volume of the substance solutions weredistributed into each well of a microtiter plate (96 wells, roundbottom). Such a procedure eventually allowed for an "in vitro"antimycotic activity assay utilizing final fungal inocula ofapproximately 5.0×10⁴ CFU/ml.

MIC evaluation

After incubating the microtiter plates for 48 hours at 35° C., the MICfor each strain was determined, which is defined as the lowestconcentration of the test substance where no visible fungal growthoccurs.

MFC evaluation

The minimal fungicidal concentration was evaluated by spot-transferringonto Petri dishes, containing Sabouraud agar, 0.01-ml volumes of all thesamples showing no visible fungal growth.

After incubation for 48 hours at 35° C., the MFC was determined, whichis defined as the lowest concentration of the test substance that doesnot allow growth of agar subcultures.

Results

The MIC values, determined after incubating samples for 48 hours at 35°C., and illustrated in Table 1-6, are exactely equal to MFC values (datanot shown), proving that these substances possess a lytic and not asimple antiproliferative activity against the tested strains.

                  TABLE 1                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of                                  5 Isovaleryl L-Carnitine esters for yeast-like fungi.                                         ST      ST     ST   ST   ST                                   Strains         1032    722    1037 1033 1001                                 ______________________________________                                        Candida albicans (562)                                                                        n.d.    3.12   1.56 1.56 3.12                                 Candida albicans (A 54)                                                                       n.d.    3.12   1.56 1.56 3.12                                 Candida albicans                                                                              n.d.    3.12   1.56 3.12 6.25                                 (ATCC 2091)                                                                   Candida albicans (Ca 2)                                                                       n.d.    1.56   1.56 n.d. 3.12                                 Candida albicans (PG)                                                                         25      3.12   3.12 3.12 6.25                                 Candida albicans (ISS 1)                                                                      25      1.56   3.12 1.56 12.5                                 Candida glabrata (ISS 1)                                                                      n.d.    6.25   3.12 3.12 6.25                                 Candida guilliermondii                                                                        n.d.    6.25   6.25 6.25 6.25                                 (ISS 1)                                                                       Candida krusei (ISS 1)                                                                        n.d.    3.12   1.56 3.12 6.25                                 Candida parapsilosis (ISS 1)                                                                  n.d.    6.25   3.12 3.12 6.25                                 Candida pseudotropicalis                                                                      n.d.    6.25   6.25 3.12 12.5                                 (EC 1)                                                                        Candida tropicalis (5705)                                                                     n.d.    3.12   3.12 3.12 6.25                                 Candida tropicalis (ISS 1)                                                                      12.5  3.12   3.12 3.12 12.5                                 Cryptococcus neoformans                                                                       n.d.    1.56   0.78 0.78 1.56                                 (4711)                                                                        Cryptococcus neoformans                                                                       50      3.12   1.56 1.56 3.12                                 (ISS 1)                                                                       Saccharomyces cerevisiae                                                                      100     3.12   3.12 3.12 6.25                                 (ATCC 7752)                                                                   Torulopsis candida (D.S.)                                                                     n.d.    6.25   3.12 3.12 6.25                                 ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 2                                                         ______________________________________                                        Mean MIC values (mcg/ml) of                                                   5 Isovaleryl L-Carnitine esters for yeast-like fungi.                                   Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 1032   42.50         5      0                                              ST 722    3.76         17      0                                              ST 1037   2.80         17      0                                              ST 1033   2.70         17      0                                              ST 1001   6.24         17      0                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

                  TABLE 3                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of                                  6 Undecyl L-Carnitine esters for yeast-like fungi.                                        ST      ST     ST   ST   ST    ST                                 Strains     712     1034   1036 1000 982   983                                ______________________________________                                        Candida albicans                                                                          12.5    1.56   1.56 n.d. n.d.  n.d.                               (562)                                                                         Candida albicans                                                                          12.5    1.56   1.56 6.25 >100  >100                               (A 54)                                                                        Candida albicans                                                                          12.5    1.56   1.56 n.d. n.d.  n.d.                               (ATCC 2091)                                                                   Candida albicans                                                                           6.25   1.56   1.56 1.56 >100  >100                               (Ca 2)                                                                        Candida albicans                                                                          12.5    3.12   3.12 n.d. n.d.  n.d.                               (PG)                                                                          Candida albicans                                                                          n.d.    1.56   1.56 3.12 n.d.  n.d.                               (ISS 1)                                                                       Candida glabrata                                                                          n.d.    3.12   3.12 6.25 n.d.  n.d.                               (ISS 1)                                                                       Candida     n.d.    6.25   6.25 6.25 n.d.  n.d.                               guilliermondii                                                                (ISS 1)                                                                       Candida krusei                                                                            n.d.    1.56   1.56 1.56 n.d.  n.d.                               (ISS 1)                                                                       Candida parapsilosis                                                                      n.d.    6.25   6.25 6.25 n.d.  n.d.                               (ISS 1)                                                                       Candida     12.5    6.25   6.25 n.d. n.d.  n.d.                               pseudotropicalis                                                              (EC 1)                                                                        Candida tropicalis                                                                         3.12   3.12   3.12 nd.  >100  >100                               (5705)                                                                        Candida tropicalis                                                                        n.d.    3.12   3.12 3.12 n.d.  n.d.                               (ISS 1)                                                                       Cryptococcus                                                                               3.12   1.56   1.56 1.56 >100  >100                               neoformans (4711)                                                             Cryptococcus                                                                              n.d.    3.12   3.12 n.d. n.d.  n.d.                               neoformans (ISS 1)                                                            Saccharomyces                                                                              6.25   3.12   3.12 n.d. n.d.  n.d.                               cerevisiae                                                                    (ATCC 7752)                                                                   Torulopsis candida                                                                        12.5    3.12   3.12 6.25 >100  >100                               (D.S.)                                                                        ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 4                                                         ______________________________________                                        Mean MIC values (mcg/ml) of                                                   6 Undecyl L-Carnitine esters for yeast-like fungi.                                      Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 712    9.37         10      0                                              ST 1034   3.03         17      0                                              ST 1036   2.84         17      0                                              ST 1000   4.21         10      0                                              ST 982    >100          5      5                                              ST 983    >100          5      5                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

                  TABLE 5                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of                                  5 L-Carnitine esters for yeast-like fungi.                                                     ST     ST     ST   ST   ST                                   Strains          1038   1052   1051 1050 1053                                 ______________________________________                                        Candida albicans (562)                                                                         3.12   3.12   3.12 6.25 12.5                                 Candida albicans (A 54)                                                                        3.12   n.d.   n.d. n.d. n.d.                                 Candida albicans (ATCC 2091)                                                                   3.12   n.d.   n.d. n.d. n.d.                                 Candida albicans (Ca 2)                                                                        1.56   1.56   1.56 3.12 3.12                                 Candida albicans (PG)                                                                          6.25   n.d.   n.d. n.d. n.d.                                 Candida albicans (ISS 1)                                                                       6.25   n.d.   n.d. n.d. n.d.                                 Candida glabrata (ISS 1)                                                                       6.25   n.d.   n.d. n.d. n.d.                                 Candida guilliermondii (ISS 1)                                                                 6.25   n.d.   n.d. n.d. n.d.                                 Candida krusei (ISS 1)                                                                         3.12   3.12   3.12 3.12 3.12                                 Candida parapsilosis (ISS 1)                                                                   6.25   n.d.   n.d. n.d. n.d.                                 Candida pseudotropicalis                                                                       6.25   n.d.   n.d. n.d. n.d.                                 (EC 1)                                                                        Candida tropicalis (5705)                                                                      3.12   3.12   3.12 6.25 6.25                                 Candida tropicalis (ISS 1)                                                                     3.12   n.d.   n.d. n.d. n.d.                                 Cryptococcus neoformans (4711)                                                                 1.56   n.d.   n.d. n.d. n.d.                                 Cryptococcus neoformans                                                                        3.12   n.d.   n.d. n.d. n.d.                                 (ISS 1)                                                                       Saccharomyces cerevisiae                                                                       6.25   3.12   3.12 3.12 3.12                                 (ATCC 7752)                                                                   Torulopsis candida (D.S.)                                                                      6.25   n.d.   n.d. n.d. n.d.                                 ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 6                                                         ______________________________________                                        Mean MIC values (mcg/ml) of                                                   5 L-Carnitine esters for yeast-like fungi.                                              Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 1038   4.40         17      0                                              ST 1052   2.80         5       0                                              ST 1051   2.80         5       0                                              ST 1050   4.37         5       0                                              ST 1053   5.62         5       0                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

Evaluation of Minimal Inhibitory Concentration (MIC) of 16 newsubstances for filamentous fungi. Experimental procedure

The following strains were used (the number of strains is in brackets):Aspergillus (5); Fusarium (1); Mucor (1); Penicillium (1).

The tested substances were: ST 712, ST 722, ST 982, ST 983, ST 1000, ST1001, ST 1032, ST 1033, ST 1034, ST 1036, ST 1037, ST 1038, ST 1050, ST1051, ST 1052, ST 1053. The assay medium was the same as the one used inthe previous study. The conidia from 72-hour cultures in Sabouraud agar,collected by washing with Sabouraud medium and Tween 80 (one drop ofTween per 10 ml of medium), were utilized as inoculum (5.0×10⁴ CFU/ml).

The assay was performed using the same procedure as described foryeast-like fungi.

Also the evaluation of MIC and MFC was as reported previously.

Results

The MIC values, reported in Tables 7-12, were exactly the same as theMFC values (data not shown), determined by a subculture tecnique.

                  TABLE 7                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of                                  5 Isovaleryl L-Carnitine esters for filamentous fungi.                                       ST      ST     ST   ST   ST                                    Strains        1032    722    1037 1033 1001                                  ______________________________________                                        Aspergillus fumigatus                                                                        >100    12.5   6.25 3.12 6.25                                  (ATCC 28212)                                                                  Aspergillus fumigatus                                                                        n.d.    12.5   6.25 3.12 6.25                                  (1/A)                                                                         Aspergillus fumigatus                                                                        n.d.    12.5   6.25 3.12 6.25                                  (2/A)                                                                         Aspergillus fumigatus                                                                        n.d.    12.5   6.25 3.12 6.25                                  (C/1)                                                                         Aspergillus niger                                                                            >100    12.5   6.25 6.25 12.5                                  (ATCC 16404)                                                                  Mucor mucedo   >100    50     12.5 6.25 >100                                  (ATCC 7941)                                                                   Fusarium sp. (ISS 1)                                                                          100    6.25   3.12 3.12 12.5                                  Penicillium sp. (1302)                                                                        100    12.5   6.25 3.12 6.25                                  ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 8                                                         ______________________________________                                        Mean MIC values (mcg/ml) of                                                   5 Isovaleryl L-Carnitine esters for filamentous fungi.                                  Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 1032   100          5       3                                              ST 722    16.40        8       0                                              ST 1037   6.64         8       0                                              ST 1033   3.90         8       0                                              ST 1001   7.03         8       1                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

                  TABLE 9                                                         ______________________________________                                        Mean MIC values (mcg/ml) of                                                   6 Undecyl L-Carnitine esters for filamentous fungi.                                       ST     ST      ST   ST   ST    ST                                 Strains     712    1034    1036 1000 982   983                                ______________________________________                                        Aspergillus 25     6.25    3.12 3.12 >100  >100                               fumigatus                                                                     (ATCC 28212)                                                                  Aspergillus 25     6.25    6.25 n.d. n.d.  n.d.                               fumigatus (1/A)                                                               Aspergillus 25     3.12    6.25 n.d. n.d.  n.d.                               fumigatus (2/A)                                                               Aspergillus 25     6.25    3.12 n.d. n.d.  n.d.                               fumigatus (C/1)                                                               Aspergillus niger                                                                         50     6.25    3.12 3.12 >100  >100                               (ATCC 16404)                                                                  Mucor mucedo                                                                              50     12.5    6.25 6.25 >100  >100                               (ATCC 7941)                                                                   Fusarium sp.                                                                              n.d.   3.12    3.12 3.12 >100  >100                               (ISS 1)                                                                       Penicillium sp.                                                                           n.d.   3.12    3.12 3.12 >100  >100                               (1302)                                                                        ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 10                                                        ______________________________________                                        Mean MIC values (mcg/ml) of                                                   6 Undecyl L-Carnitine esters for filamentous fungi.                                     Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 712    30.00        6       1                                              ST 1034   5.85         8       0                                              ST 1036   4.29         8       0                                              ST 1000   3.76         5       0                                              ST 982    >100         5       5                                              ST 983    >100         5       5                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

                  TABLE 11                                                        ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of                                  5 L-Carnitine esters for filamentous fungi.                                                  ST      ST     ST    ST   ST                                   Strains        1038    1052   1050  1051 1053                                 ______________________________________                                        Aspergillus fumigatus                                                                        12.5    3.12   6.25  3.12 6.25                                 (ATCC 28212)                                                                  Aspergillus fumigatus                                                                        12.5    n.d.   n.d.  n.d. n.d.                                 (1/A)                                                                         Aspergillus fumigatus                                                                        12.5    n.d.   n.d.  n.d. n.d.                                 (2/A)                                                                         Aspergillus fumigatus                                                                         6.25   n.d.   n.d.  n.d. n.d.                                 (C/1)                                                                         Aspergillus niger                                                                            12.5    3.12   6.25  6.25 3.12                                 (ATCC 16404)                                                                  Mucor mucedo   25      6.25   12.5  6.25 12.5                                 (ATCC 7941)                                                                   Fusarium sp. (ISS 1)                                                                          6.25   6.25   6.25  6.25 6.25                                 Penicillium sp. (1302)                                                                       12.5    3.12   3.12  3.12 3.12                                 ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 12                                                        ______________________________________                                        Mean MIC values (mcg/ml) of                                                   5 L-Carnitine esters for filamentous fungi.                                             Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 1038   12.50        8       0                                              ST 1052   4.37         5       0                                              ST 1050   6.85         5       0                                              ST 1051   6.87         5       0                                              ST 1053   6.24         5       0                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

Evaluation of Minimal Inhibitory Concentration (MIC) of 7 new substancesfor dermatophytic fungi. Experimental procedure

The following strains were used: Trichophyton menthagrophytes ATCC 9533,Trichophyton rubrum IHEM 4274, Microsporum canis IHEM 3522,Epydermophyton floccosum IHEM 3495. The tested substances were: ST 722,ST 1033, ST 1037, ST 1050, ST 1051, ST 1052,. ST 1053.

The assay was performed following the same procedure described for theyeasts, by using 7-day fungi cultures grown at 30° C. in Potato Dextroseagar, and harvested with Sabouraud broth and Tween 80 (0.5%).

After washing with Yeast Nitrogen Base, supplemented with asparagine andglucose (as carbon source), the fungal suspension was fragmented using atissue grinder until the largest clumps or aggregates were undetectablewithin the grinder. This fungal suspension was then appropriatelydiluted in YNB to yield a concentration of 2.0×10⁵ infectiveparticles/ml (small mycelium fragments, micro-and macroaleuriospores).

Volumes of 100 μl both of the fungal suspensions and substancesolutions, two-fold serially diluted in assay medium, were distributedinto each well of a microtiter plate (96 wells, round bottom), which wasincubated at 35° C. for 96 hours.

The MIC values, substantially corresponding to the MFC values (data notshown), are reported in Table 13-14.

                  TABLE 13                                                        ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of                                  7 new substances for dermatophytic fungi.                                                 ST     ST     ST   ST   ST   ST   ST                              Strains     722    1037   1033 1052 1050 1051 1053                            ______________________________________                                        Epidermophyton                                                                            50     25     25   25   50   25   50                              floccosum                                                                     (IHEM 3495)                                                                   Microsporum canis                                                                         25     12.5   6.25 6.25 12.5 12.5 6.25                            (IHEM 3522)                                                                   Trichophyton rubrum                                                                       12.5   6.25   6.25 6.25 6.25 6.25 6.25                            (IHEM 4274)                                                                   Trichophyton                                                                              25     12.5   12.5 12.5 12.5 12.5 6.25                            menthagrophites                                                               (ATCC 9533)                                                                   ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Mean MIC values (mcg/ml) of                                                   7 new substances for dermatophytic fungi.                                               Mean         Tested  Resistant strains                              Compound  MIC values*  strains (MIC > 100)                                    ______________________________________                                        ST 722    28.12        4       0                                              ST 1037   14.06        4       0                                              ST 1033   12.50        4       0                                              ST 1052   12.50        4       0                                              ST 1050   20.31        4       0                                              ST 1051   14.06        4       0                                              ST 1053   14.06        4       0                                              ______________________________________                                         * = Mean MIC values versus susceptible strains.                          

Evaluation of the protective effects of ST 722 in an experimentalsubcutaneous infection with Candida albicans in mice. Experimentalprocedure Animals

Male CD₁ outbred mice (IFFA Credo) aged 8 weeks were used (4 animals pergroup).

Bacterial strain

A pathogenic strain of Candida was used, namely C. albicans ISS 1. TheMIC of ST 722 against this strain was 1.56 mcg/ml, whereas the MIC ofMiconazole, a reference compound, was not determined, although the dataavailable from the literature indicate MIC values raging from 0.5 to 10mcg/mi.

Compounds

The infected animals were treated with ST 722 dissolved in saline, andMiconazole diluted in saline from a stock solution containing 1%Dimethyl Formamide.

Infective model

From an overnight broth culture of C. albicans in Sabouraud broth, afungal suspension was prepared in sterile saline containing 5.0×10⁵cells/mi. An inoculum of 0.5 ml of this suspension was injectedsubcutaneously into the abdominal wall of each animal.

The compounds were then administered by subcutaneous injection in thesame area of the fungal challenge at the dose of 50 mcg (in 0.2 ml ofsaline) immediately after the inoculum and following 48 and 96 hours(150 total mcg per animal).

Then, 48 hours following the last treatment (6 days after the challengewith Candida), the animals were sacrificed, and the abdominal wall aswell as the peritoneum were dissected out.

The excised tissues were then homogenized in 5 ml of sterile saline witha Potter-Elvehjem tissue grinder. The resulting suspensions,appropriately diluted, where plated onto Petri dishes containing aselective agar medium (BIGGY agar), which allows to easily detect theyeast Colony Forming Units (CFU/ml).

Evaluation of results

The number of colonies scored in a series of dilutions for each singlesample consents to calculate the number of yeast cells present in theinfected tissue sample as follows: ##EQU1## Where Z_(i) is the number ofdilutions performed, N_(i) is the number of plates prepared for eachdilution, and C_(i) is the total number of yeast cells scored in eachdilution.

Results

The results reported in Table 1 show that ST 722, administered asdescribed above, substantially inhibits the infection with Candida,inducing the disappearance of Candida cells from the abdomen of treatedanimals (3 sterile samples out of 4). Similar experimental results wereattained with Miconazole.

                  TABLE 1                                                         ______________________________________                                        Effect of ST 722 and Miconazole (reference compound) in an ex-                perimental subcutaneous infection with Candida albicans in mice.              Treatment      Sample  CFU/mouse                                              ______________________________________                                        Control        1-4     .sup.   1.19 × 10.sup.4 (*)                      ST 722         1       sterile                                                               2       sterile                                                               3       sterile                                                               4       1.36 × 10.sup.2                                  Miconazole     1       sterile                                                               2       sterile                                                               3       3.60 × 10.sup.1                                                 4       8.10 × 10.sup.1                                  ______________________________________                                         (*) = Mean CFU value of 4 control samples.                               

Evaluation of the protective effect of ST 722 in a systemic experimentalinfection with Candida albicans in mice. Experimental procedure Animal

Male CD1 mice (C. River) aged 6 or 13 weeks were used (12-15 animals pergroup).

Yeast strain

The pathogenic strain of Candida utilized, i.e. C. albicans PG, wasable, when inoculated intravenously, to cause multiple renal lesions andsystemic dissemination, provoking death of the animals within 40 daysfrom the challenge. The compound ST 722 exibits against this Candidastrain a value of MIC and MFC equal to 3.12 mcg/ml.

Inoculum preparation

A broth culture in Sabouraud broth is prepared from a 48-hour agar slantculture.

After incubation for 48 hours at 37° C., the broth culture is firstwashed 3 times with sterile saline, then adjusted to a concentration of2.0×10⁶ cells/ml. Volumes of 0.25 ml of this suspension are i.v.injected into the animals, with a resulting infective dose approximatelycorresponding to DL100.

Treatment

ST 722 was administered per os following two different protocols:

I) 100 mg/kg administered at +2, +12, +24, +36 and +48 hours (i.e. 5administrations in two days) with respect to the challenge.

II) 100 mg/kg administered twice daily on 6 consecutive days (i.e. 12adimistrations) starting from day -2 to day +3 with respect to the dayof inoculation (day 0).

Evaluation criteria

After the challenge, the animals were followed up for 60 days, and thepercent mortality as well as the mean survival time (MST) wereevaluated. The statistical significance of the results was evaluated bymeans of Fisher "exact test" and Mann-Withney "U" test, respectively.

Results

The protective effect of ST 722. only in terms of MST prolongation, wasconfirmed in both protocol I and II. In fact, also in protocol II thepercent mortality of mice was completely unaffected, although the numberof administrations was raised and the ST 722 treatment was started twodays prior to the challenge (Table 2 and 3 ).

                  TABLE 2                                                         ______________________________________                                        Protective effect of ST 722 in mice experimentally                            infected with a C. albicans pathogenic strain.                                Treatment                                                                              Dead /Total                                                                              % Mortality  MST (days).sup.a                             ______________________________________                                        Control  13/15      86.6         22 (15-30)                                   ST 722.sup.b                                                                           10/14      71.4         37 (25-≧60)                           ______________________________________                                         .sup.a = Mean survival time (range of variation is in brackets). The          animals were followed up for 60 consecutive days after challenge (day 0).     .sup.b = The substance was administered according to protocol I.                = p ≦ 0.05 (MannWithney "U" test).                              

                  TABLE 3                                                         ______________________________________                                        Protective effect of ST 722 in mice experimentally                            infected with a C. albicans pathogenic strain.                                Treatment                                                                              Dead/Total % Mortality  MST (days).sup.a                             ______________________________________                                        Control  10/12      83.3         26 (22-39)                                   ST 722.sup.b                                                                           10/14      71.4         44 (32-≧60)                           ______________________________________                                         .sup.a = Mean survival time (range of variation is in brackets). The          animals were followed up for 60 consecutive days after challenge (day 0).     .sup.b = The substance was administered according to protocol II.               = p ≦ 0.05 (MannWithney "U" test).                              

We claim:
 1. The method for treating a mycosis in a human or animalwhich comprises administering to the human or animal an antimycoticeffective amount of an ester of acyl L-carnitine of the general formula,##STR8## wherein: R is an straight or branched saturated aliphatichydrocarbon carboxylic acyl group having 2-16 carbon atoms,n is aninteger from 7 to 15, and X is the anion of a pharmacologicallyacceptable acid.
 2. The method of claim 1, wherein the mycosis is aninfection by a yeast.
 3. The method of claim 2, wherein the mycosis isan infection sustained by Candida albicans, Candida tropicalis, Candidapseudotropicalis, Torulopsis candida, Saccharomices cerevisiae orCryptococcus neoformans.
 4. The method of claim 1, wherein the mycosisis an infection by a filamentous fungus.
 5. The method of claim 4,wherein the mycosis is an infection sustained by Aspergillus fumigatus,Aspergillus niger, or Mucor mucedo.
 6. The method of claim 1 wherein Ris isobutyryl or isovaleryl.
 7. The method of claim 1 wherein n is 10.8. The method of claim 1 wherein X is selected from: chloride; bromide;iodide; acid aspartate; acid citrate; tartrate; acid phosphate; acidfumarate; glycerophosphate; glucosephosphate; lactate; acid maleate;orotate; acid oxalate; acid sulphate; trichloroacetate, trifluoroacetateand methansulphonate.
 9. The method of claim 1 wherein the ester isisovalery L-carnitine undecyl ester chloride.
 10. The method of claim 1wherein the ester is isobutyryl L-carnitine undecyl ester chloride.